LOCUS       sequence-368334-       10577 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression and purification of N-terminal His-MBP tagged SuperFi 
            Cas9 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10577)
  AUTHORS   Bravo JPK, Liu MS, Hibshman GN, Dangerfield TL, Jung K, McCool RS, 
            Johnson KA, Taylor DW
  TITLE     Structural basis for mismatch surveillance by CRISPR-Cas9.
  JOURNAL   Nature. 2022 Mar 2. pii: 10.1038/s41586-022-04470-1. doi: 
            10.1038/s41586-022-04470-1.
  PUBMED    35236982
REFERENCE   2  (bases 1 to 10577)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2022 Mar 2. pii: 10.1038/s41586-022-04470-1. doi: 
            10.1038/s41586-022-04470-1."
COMMENT     Sequence Label: pDT-SuperFi-Cas9
FEATURES             Location/Qualifiers
     source          1..10577
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             769..960
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(954..976)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1062..1201
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1117..1136
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1216..1233)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1387..1975)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1467..1486)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(2086..2105)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             2097..2912
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             2097..2912
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2170..2189)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(3004..3459)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3141..3162)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3353..3372
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3496..3543)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3539..3557
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3610..3627)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(3692..7798)
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVDGDDKVDDDDKMIAKSEDEIGDATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             complement(7808..7828)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(7859..8956)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(7862..7885)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     CDS             complement(8960..8977)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             8988..9010
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    9025..9049
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(9049..9068)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(9050..9068)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(9152..9171)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        9377..9454
                     /gene="lacI"
                     /label=lacI promoter
     CDS             9455..10537
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(9474..9493)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 ccgggatctc gaccgatgcc cttgagagcc ttcaacccag tcagctcctt ccggtgggcg
       61 cggggcatga ctatcgtcgc cgcacttatg actgtcttct ttatcatgca actcgtagga
      121 caggtgccgg cagcgctctg ggtcattttc ggcgaggacc gctttcgctg gagcgcgacg
      181 atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg ccctcgctca agccttcgtc
      241 actggtcccg ccaccaaacg tttcggcgag aagcaggcca ttatcgccgg catggcggcc
      301 ccacgggtgc gcatgatcgt gctcctgtcg ttgaggaccc ggctaggctg gcggggttgc
      361 cttactggtt agcagaatga atcaccgata cgcgagcgaa cgtgaagcga ctgctgctgc
      421 aaaacgtctg cgacctgagc aacaacatga atggtcttcg gtttccgtgt ttcgtaaagt
      481 ctggaaacgc ggaagtcagc gccctgcacc attatgttcc ggatctgcat cgcaggatgc
      541 tgctggctac cctgtggaac acctacatct gtattaacga agcgctggca ttgaccctga
      601 gtgatttttc tctggtcccg ccgcatccat accgccagtt gtttaccctc acaacgttcc
      661 agtaaccggg catgttcatc atcagtaacc cgtatcgtga gcatcctctc tcgtttcatc
      721 ggtatcatta cccccatgaa cagaaatccc ccttacacgg aggcatcagt gaccaaacag
      781 gaaaaaaccg cccttaacat ggcccgcttt atcagaagcc agacattaac gcttctggag
      841 aaactcaacg agctggacgc ggatgaacag gcagacatct gtgaatcgct tcacgaccac
      901 gctgatgagc tttaccgcag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
      961 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     1021 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     1081 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     1141 gagtgcacca ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag
     1201 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     1261 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     1321 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     1381 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     1441 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1501 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1561 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1621 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1681 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1741 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1801 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     1861 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     1921 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1981 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     2041 tttggtcatg aacaataaaa ctgtctgctt acataaacag taatacaagg ggtgttatga
     2101 gccatattca acgggaaacg tcttgctcta ggccgcgatt aaattccaac atggatgctg
     2161 atttatatgg gtataaatgg gctcgcgata atgtcgggca atcaggtgcg acaatctatc
     2221 gattgtatgg gaagcccgat gcgccagagt tgtttctgaa acatggcaaa ggtagcgttg
     2281 ccaatgatgt tacagatgag atggtcagac taaactggct gacggaattt atgcctcttc
     2341 cgaccatcaa gcattttatc cgtactcctg atgatgcatg gttactcacc actgcgatcc
     2401 ccgggaaaac agcattccag gtattagaag aatatcctga ttcaggtgaa aatattgttg
     2461 atgcgctggc agtgttcctg cgccggttgc attcgattcc tgtttgtaat tgtcctttta
     2521 acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg aatgaataac ggtttggttg
     2581 atgcgagtga ttttgatgac gagcgtaatg gctggcctgt tgaacaagtc tggaaagaaa
     2641 tgcataaact tttgccattc tcaccggatt cagtcgtcac tcatggtgat ttctcacttg
     2701 ataaccttat ttttgacgag gggaaattaa taggttgtat tgatgttgga cgagtcggaa
     2761 tcgcagaccg ataccaggat cttgccatcc tatggaactg cctcggtgag ttttctcctt
     2821 cattacagaa acggcttttt caaaaatatg gtattgataa tcctgatatg aataaattgc
     2881 agtttcattt gatgctcgat gagtttttct aagaattaat tcatgagcgg atacatattt
     2941 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     3001 cctaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct
     3061 cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg
     3121 agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact
     3181 ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac
     3241 cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga
     3301 gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga
     3361 aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca
     3421 ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc ccattcgcca atccggatat
     3481 agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa ggggttatgc
     3541 tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt tgttagcagc
     3601 cggatctcag tggtggtggt ggtggtgctc gagtgcggcc gcaagcttgt cgacggagct
     3661 cgaattcgga tcctgcaaag caccggcctc gttagtcacc tcctagctga ctcaaatcaa
     3721 tgcgtgtttc ataaagacca gtgatggatt gatggataag agtggcatct aaaacttctt
     3781 ttgtagacgt atatcgttta cgatcaattg ttgtatcaaa atatttaaaa gcagcgggag
     3841 ctccaagatt cgtcaacgta aataaatgaa taatattttc tgcttgttca cgtattggtt
     3901 tgtctctatg tttgttatat gcactaagaa ctttatctaa attggcatct gctaaaataa
     3961 cacgcttaga aaattcactg atttgctcaa taatctcatc taaataatgc ttatgctgct
     4021 ccacaaacaa ttgtttttgt tcgttatctt ctggactacc cttcaacttt tcataatgac
     4081 tagctaaata taaaaaattc acatatttgc ttggcagagc cagctcattt cctttttgta
     4141 attctccggc actagccagc atccgtttac gaccgttttc taactcaaaa agactatatt
     4201 taggtagttt aatgattaag tcttttttaa cttccttata tcctttagct tctaaaaagt
     4261 caatcggatt tttttcaaag gaacttcttt ccataattgt gatccctagt aactctttaa
     4321 cggattttaa cttcttcgat ttcccttttt ccaccttagc aaccactagg actgaataag
     4381 ctaccgttgg actatcaaaa ccaccatatt tttttggatc ccagtctttt ttacgagcaa
     4441 taagcttgtc cgaatttctt tttggtaaaa ttgactcctt ggagaatccg cctgtctgta
     4501 cttctgtttt cttgacaata ttgacttggg gcatggacaa tactttgcgc actgtggcaa
     4561 aatctcgccc tttatcccag acaatttctc cagtttcccc attagtttcg attagagggc
     4621 gtttgcgaat ctctccattt gcaagtgtaa tttctgtttt gaagaagttc atgatattag
     4681 agtaaaagaa atattttgcg gttgcatcgc ctatttcatc ctcagactta gcaatcattt
     4741 tatcatcatc atcaacttta tcatcaccat cgacaaactc cgattcaagt tttggatatt
     4801 tcttaatcaa agcagttcca acgacggcat ttagatacgc atcatgggca tgatggtaat
     4861 tgttaatctc acgtacttta tagaattgga aatcttttcg gaagtcagaa actaatttag
     4921 attttaaggt aatcacttta acctctcgaa taagtttatc attttcatcg tatttagtat
     4981 tcatgcgact atccaaaatt tgtgccacat gcttagtgat ttggcgagtt tcaaccaatt
     5041 ggcgtttgat aaaaccagct ttatcaagtt cactcaaacc tccacgttca gctttcgtta
     5101 aattatcaaa cttacgttga gtgattaact tggcgtttag aagttgtctc caatagtttt
     5161 tcatcttttt gactacttct tcacttggaa cgttatccga tttaccacga tttttatcag
     5221 aacgcgttaa gaccttattg tctattgaat cgtctttaag gaaactttgt ggaacaatgt
     5281 gatcgacatc ataatcactt aaacgattaa tatctaattc ttggtccaca tacatgtctc
     5341 ttccattttg gagataatag agatagagct tttcattttg caattgagta ttttcaacag
     5401 gatgctcttt aagaatctga cttcctaatt ctttgatacc ttcttcgatt cgtttcatac
     5461 gctctcgcga atttttctgg cccttttgag ttgtctgatt ttcacgtgcc atttcaataa
     5521 cgatattttc tggcttatgc cgccccatta ctttgaccaa ttcatcaaca acttttacag
     5581 tctgtaaaat acctttttta atagcagggc taccagctaa atttgcaata tgttcatgta
     5641 aactatcgcc ttgtccagac acttgtgctt tttgaatgtc ttctttaaat gtcaaactat
     5701 catcatggat cagctgcata aaattgcgat tggcaaaacc atctgatttc aaaaaatcta
     5761 atattgtttt gccagattgc ttatccctaa taccattaat caattttcga gacaaacgtc
     5821 cccaaccagt ataacggcga cgtttaagct gtttcatcac cttatcatca aagaggtgag
     5881 catatgtttt aagtctttcc tcaatcatct ccctatcttc aaataaggtc aatgttaaaa
     5941 caatatcctc taagatatct tcattttctt cattatccaa aaaatcttta tctttaataa
     6001 tttttagcaa atcatggtag gtacctaatg aagcattaaa tctatcttca actcctgaaa
     6061 tttcaacact atcaaaacat tctatttttt tgaaataatc ttcttttaat tgcttaacgg
     6121 ttacttttcg atttgttttg aagagtaaat caacaatggc tttcttctgt tcacctgaaa
     6181 gaaatgctgg ttttcgcatt ccttcagtaa catatttgac ctttgtcaat tcgttataaa
     6241 ccgtaaaata ctcataaagc aaactatgtt ttggtagtac tttttcattt ggaagatttt
     6301 tatcaaagtt tgtcatgcgt tcaataaatg attgagctga agcaccttta tcgacaactt
     6361 cttcaaaatt ccatggggta attgtttctt cagacttccg agtcatccat gcaaaacgac
     6421 tattgccacg cgccaatgga ccaacataat aaggaattcg aaaagtcaag attttttcaa
     6481 tcttctcacg attgtctttt aaaaatggat aaaagtcttc ttgtcttctc aaaatagcat
     6541 gcagctcacc caagtgaatt tgatggggaa tagagccgtt gtcaaaggtc cgttgcttgc
     6601 gcagcaaatc ttcacgattt agtttcacca ataattcctc agtaccatcc attttttcta
     6661 aaattggttt gataaattta taaaattctt cttggctagc tcccccatca atataacctg
     6721 catatccgtt ttttgattga tcaaaaaaga tttctttata cttttctgga agttgttgtc
     6781 gaactaaagc ttttaaaaga gtcaagtctt gatgatgttc atcgtagcgt ttaatcattg
     6841 aagctgatag gggagcctta gttatttcag tatttactct taggatatct gaaagtaaaa
     6901 tagcatctga taaattctta gctgccaaaa acaaatcagc atattgatct ccaatttgcg
     6961 ccaataaatt atctaaatca tcatcgtaag tatcttttga aagctgtaat ttagcatctt
     7021 ctgccaaatc aaaatttgat ttaaaattag gggtcaaacc caatgacaaa gcaatgagat
     7081 tcccaaataa gccatttttc ttctcaccgg ggagctgagc aatgagattt tctaatcgtc
     7141 ttgatttact caatcgtgca gaaagaatcg ctttagcatc tactccactt gcgttaatag
     7201 ggttttcttc aaataattga ttgtaggttt gtaccaactg gataaatagt ttgtccacat
     7261 cactattatc aggatttaaa tctccctcaa tcaaaaaatg accacgaaac ttaatcatat
     7321 gcgctaaggc caaatagatt aagcgcaaat ccgctttatc agtagaatct accaattttt
     7381 ttcgcagatg atagatagtt ggatatttct catgataagc aacttcatct actatatttc
     7441 caaaaatagg atgacgttca tgcttcttgt cttcttccac caaaaaagac tcttcaagtc
     7501 gatgaaagaa actatcatct actttcgcca tctcatttga aaaaatctcc tgtagataac
     7561 aaatacgatt cttccgacgt gtataccttc tacgagctgt ccgtttgaga cgagtcgctt
     7621 ccgctgtctc tccactgtca aataaaagag cccctataag attttttttg atactgtggc
     7681 ggtctgtatt tcccagaacc ttgaactttt tagacggaac cttatattca tcagtgatca
     7741 ccgcccatcc gacgctattt gtgccgatat ctaagcctat tgagtatttc ttatccatcg
     7801 aggctgctcc ctggaaatac aggttttcac tagtgttgtt gttgttcgag ctcgaattag
     7861 tctgcgcgtc tttcagggct tcatcgacag tctgacgacc gctggcggcg ttgatcaccg
     7921 cagtacgcac ggcataccag aaagcggaca tctgcgggat gttcggcatg atttcacctt
     7981 tctgggcgtt ttccatagtg gcggcaatac gtggatcttt cgccaactct tcctcgtaag
     8041 acttcagcgc tacggcaccc agcggtttgt ctttattaac cgcttccaga ccttcatcag
     8101 tcagcagata gttttcgagg aactcttttg ccagctcttt gttcggactg gcggcgttaa
     8161 tacctgcgct cagcacgcca acgaacggtt tggatggttg acccttgaag gtcggcagta
     8221 ccgttacacc ataattcact ttgctggtgt cgatgttgga ccatgcccac gggccgttga
     8281 tggtcatcgc tgtttcgcct ttattaaagg cagcttctgc gatggagtaa tcggtgtctg
     8341 cattcatgtg tttgttttta atcaggtcaa ccaggaaggt cagacccgct ttcgcgccag
     8401 cgttatccac gcccacgtct ttaatgtcgt acttgccgtt ttcatacttg aacgcataac
     8461 ccccgtcagc agcaatcagc ggccaggtga agtacggttc ttgcaggttg aacatcagcg
     8521 cgctcttacc tttcgctttc agttctttat ccagcgccgg gatctcttcc caggtttttg
     8581 gcgggttcgg cagcagatct ttgttataaa tcagcgataa cgcttcaaca gcgatcgggt
     8641 aagcaatcag cttgccgttg taacgtacgg catcccaggt aaacggatac agcttgtcct
     8701 ggaacgcttt gtccggggtg atttcagcca acaggccaga ttgagcgtag ccaccaaagc
     8761 ggtcgtgtgc ccagaagata atgtcagggc catcgccagt tgccgcaacc tgtgggaatt
     8821 tctcttccag tttatccgga tgctcaacgg tgactttaat tccggtatct ttctcgaatt
     8881 tcttaccgac ttcagcgaga ccgttatagc ctttatcgcc gttaatccag attaccagtt
     8941 taccttcttc gatttttcca tggtgatggt gatggtgcat atgtatatct ccttcttaaa
     9001 gttaaacaaa attatttcta gaggggaatt gttatccgct cacaattccc ctatagtgag
     9061 tcgtattaat ttcgcgggat cgagatctcg atcctctacg ccggacgcat cgtggccggc
     9121 atcaccggcg ccacaggtgc ggttgctggc gcctatatcg ccgacatcac cgatggggaa
     9181 gatcgggctc gccacttcgg gctcatgagc gcttgtttcg gcgtgggtat ggtggcaggc
     9241 cccgtggccg ggggactgtt gggcgccatc tccttgcatg caccattcct tgcggcggcg
     9301 gtgctcaacg gcctcaacct actactgggc tgcttcctaa tgcaggagtc gcataaggga
     9361 gagcgtcgag atcccggaca ccatcgaatg gcgcaaaacc tttcgcggta tggcatgata
     9421 gcgcccggaa gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt
     9481 cgcagagtat gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca
     9541 cgtttctgcg aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc
     9601 caaccgcgtg gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc
     9661 cagtctggcc ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca
     9721 actgggtgcc agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc
     9781 ggcggtgcac aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga
     9841 tgaccaggat gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga
     9901 tgtctctgac cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact
     9961 gggcgtggag catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt
    10021 aagttctgtc tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca
    10081 aattcagccg atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac
    10141 catgcaaatg ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat
    10201 ggcgctgggc gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc
    10261 ggtagtggga tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat
    10321 caaacaggat tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca
    10381 gggccaggcg gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac
    10441 cctggcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
    10501 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtaagtt
    10561 agctcactca ttaggca
//
